Advancements in treatments for tendinopathy have been hampered, in part, because our basic understanding of the pathophysiology of tendinopathy and in general tendon mechanobiology is poor. Therefore, our long-term research goal is to develop a fundamental understanding of the mechanotransduction pathways in tenocytes that contribute to tendinopathy and the mechanotransduction pathways that contribute to tendon regeneration, towards identifying therapeutic strategies for treating tendinopathy and promoting functional healing. New evidence has given us key insights into how tendon functions under physiological loads. These micro-mechanical studies suggest that tendon sustains its loading environment by functioning as a typical fiber composite, where extension occurs through a combination of fiber sliding between adjacent collagen units and fiber extension. Thus, we can postulate that cells, situated along the fibers, are subjected to a complex loading environment encompassing varying levels of shear and tension during physiological loading. These observations have prompted us to form the central hypothesis for this research, which is local shear strains in combination with local tensile strains are key regulators of tenocyte metabolism, ultimately impacting the balance between anabolic and catabolic activity. Specific to the central hypothesis, we also hypothesize that these mechanotransduction events involve cellular Ca2+ signals, which represents one central pathway by which cells may detect and respond to their mechanical environment. Recently, we developed a synthetic fiber composite hydrogel material that captures aspects of the micromechanical behavior unique to tendons, encompassing local shear and tension. Our preliminary findings indeed point towards the importance of the local strain environment in regulating cell function. Therefore, the primary objective of this exploratory grant is to test our central hypothesis in the following three specific aims: Aim 1) Develop and characterize our new fiber composite material, optimizing methods for controlled manipulation of the micromechanics and cellular strains, which capture the micromechanics in healthy and damaged tendon. Aim 2) Define and characterize calcium signals in tenocytes in response to changes in their local mechanical environment using genetically encoded calcium sensors. Aim 3) Elucidate calcium-mediated events that direct tenocyte anabolic and catabolic activity in response to changes in their local environment. The proposed research is innovative because our new synthetic fiber composite exhibits well-controlled shear/tension ratios, the use of genetically encoded calcium sensors enables the nature of the signal to be defined in space and time, and the use of specially designed straining rigs enables in situ and real time assessment during the application of gross strains and when combined provide a unique platform for studying tenocyte mechanotransduction. Completion of these studies is expected to demonstrate that a microenvironment comprised of shear and tension regulates tenocyte metabolism and that the levels of shear/tension are critical to maintaining a healthy response. We also expect to have established a viable platform for in situ and real time tenocyte mechanotransduction research having provided new insights into Ca2+-mediated mechanotransduction events. PUBLIC HEALTH RELEVANCE: Tendon injuries represent a significant fraction of the more than thirty million musculoskeletal injuries reported each year in the US alone. However, treatment options offer imperfect solutions and this observation is partly attributed to our poor understanding of tendon biology. The proposed research aims to develop a new platform to better understand tenocyte, or tendon cell, mechanotransduction, a process thought to be key to regulating the balance between anabolic and catabolic metabolisms. By capturing the complex local strain behavior of healthy and damaged tendon in this new platform, this research aims to provide new insights into tendon mechanobiology which may help to identify better therapies for treating damaged tendons.